Project Summary Viral genome packaging is a complex, non-spontaneous, multi-step enzymatic reaction that in tailed bacteriophages and herpesviruses proceeds via formation of an empty precursor capsid (or procapsid) that is filled with genetic material by the action of two proteins, known as large and small terminase. Though commonly studied as individual subunits, viral terminases bind, pump and cleave viral DNA assembled into large macromolecular complexes of poorly characterized structure, function and composition. The recent discovery of a potent antiviral agent specific to Human Cytomegalovirus small terminase subunit (pUL56) has grown further interest in viral packaging motors. In this grant, combining hybrid methods in structural biology (i.e. X-crystallography, cryo-electron microscopy, hydrogen/deuterium exchange mass spectrometry) with modern biochemical approaches (i.e. conformation- specific synthetic Fabs, site directed mutagenesis, yeast 1-hybrid), we seek to understand the principles governing viral genome packaging through the comparative analysis of motors from different DNA viruses. We are particularly interested in deciphering the atomic structure of macromolecular assemblies formed by terminase subunits during the packaging reaction and the role of S-terminase that is functionally conserved from bacterial viruses to herpesviruses. This research tries to fill a significant and growing knowledge gap between the enzymology of genome packaging, which is increasing well-understood thanks to single molecule biophysical studies, and the molecular machines catalyzing packaging. Building upon the work initiated in the previous funding cycle, we seek to: 1.) Elucidate the architecture of terminase assemblies formed during viral genome packaging; 2.) Determine the conserved architecture of Human Cytomegalovirus small terminase (pUL56) and its interaction with viral DNA and the antiviral drug letermovir.